Molecular Prevalence of Blastocystis sp. from Patients with Diarrhea in the Republic of Korea.

Blastocystis sp. is the most common intestinal protozoan affecting human health worldwide. Several studies have reported the prevalence of Blastocystis sp. in various regions of the Republic of Korea. However, limited data are available on the prevalence and subtype (ST) distribution of this parasite among regions. Therefore, we investigated the prevalence and ST distributions of this parasite in the Republic of Korea. For this purpose, 894 stool specimens were collected from patients with diarrhea and tested for the presence of Blastocystis sp. using PCR analysis. The isolates were subsequently subtyped. The overall prevalence was 11.6%. Of the 104 isolates, ST3 was the most prevalent, followed by ST1. Additionally, a single case of the rare subtype ST8 was identified, representing the first reported case in the Republic of Korea. The results suggested that the predominance of ST3 observed in this study reflects human-to-human transmission with low genetic diversity within the ST, while ST1 transmission is likely correlated with animals. In the future, to better understand Blastocystis sp. transmission dynamics, human, animal, and environmental factors should be studied from a "One Health" perspective.

Guiding Hepatic Differentiation of Pluripotent Stem Cells Using 3D Microfluidic Co-Cultures with Human Hepatocytes.

Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate.

The association between autoantibody types and salivary gland hypofunction in patients with primary Sjögren's syndrome.

BACKGROUND: This study analyzed the association between autoantibody types and salivary gland hypofunction in patients with primary Sjögren's syndrome (pSS). METHODS: A retrospective analysis was performed on patients who visited the Department of Orofacial Pain and Oral Medicine at Yonsei University Dental Hospital from January 1, 2010 to May 31, 2021, and who were diagnosed with pSS. Out of 191 patients who fulfilled the 2016 American College of Rheumatology/European League Against Rheumatism classification criteria, 50 were positive for both anti-Ro/SSA and anti-La/SSB, whereas 97 had anti-Ro/SSA but not anti-La/SSB antibodies. Forty-four patients for whom neither anti-Ro/SSA nor anti-La/SSB antibodies were found were diagnosed with Sjögren's syndrome by minor salivary gland biopsy. RESULTS: The anti-Ro/SSA antibody-positive group showed higher rheumatoid factor (RF) levels than the anti-Ro/SSA antibody-negative group. The anti-La/SSB antibody-positive group showed lower unstimulated whole saliva (UWS), stimulated whole saliva (SWS), higher erythrocyte sedimentation rate and RF level than the anti-La/SSB antibody-negative group. In addition, the group with both anti-Ro/SSA and anti-La/SSB antibodies showed lower UWS than the group with only anti-Ro/SSA antibodies. However, there were no significant differences in UWS or SWS after taking pilocarpine, and C-reactive protein. CONCLUSIONS: UWS and SWS were lower when a patient was positive for anti-La/SSB, showing that anti-La/SSB is more likely to be involved in salivary gland hypofunction than anti-Ro/SSA in patients with pSS. Therefore, performing laboratory tests, including anti-La/SSB, helps predict the prognosis of salivary gland function in patients with suspected pSS.

Effects of the Washing Time and Washing Solution on the Biocompatibility and Mechanical Properties of 3D Printed Dental Resin Materials.

Three-dimensional (3D) printing technology is highly regarded in the field of dentistry. Three-dimensional printed resin restorations must undergo a washing process to remove residual resin on the surface after they have been manufactured. However, the effect of the use of different washing solutions and washing times on the biocompatibility of the resulting resin restorations is unclear. Therefore, we prepared 3D-printed denture teeth and crown and bridge resin, and then washed them with two washing solutions (isopropyl alcohol and tripropylene glycol monomethyl ether) using different time points (3, 5, 10, 15, 30, 60, and 90 min). After this, the cell viability, cytotoxicity, and status of human gingival fibroblasts were evaluated using confocal laser scanning. We also analyzed the flexural strength, flexural modulus, and surface SEM imaging. Increasing the washing time increased the cell viability and decreased the cytotoxicity (p < 0.001). Confocal laser scanning showed distinct differences in the morphology and number of fibroblasts. Increasing the washing time did not significantly affect the flexural strength and surface, but the flexural modulus of the 90 min washing group was 1.01 ± 0.21 GPa (mean ± standard deviation), which was lower than that of all the other groups and decreased as the washing time increased. This study confirmed that the washing time affected the biocompatibility and mechanical properties of 3D printed dental resins.

Sestrin2 Regulates Osteoclastogenesis via the p62-TRAF6 Interaction.

The receptor activator of nuclear factor-kappa B ligand (RANKL) mediates osteoclast differentiation and functions by inducing Ca2+ oscillations, activating mitogen-activated protein kinases (MAPKs), and activating nuclear factor of activated T-cells type c1 (NFATc1) via the RANK and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interaction. Reactive oxygen species (ROS) also plays an important role during osteoclastogenesis and Sestrin2, an antioxidant, maintains cellular homeostasis upon stress injury via regulation of ROS, autophagy, and inflammation. However, the role of Sestrin2 in osteoclastogenesis remains unknown. In this study, we investigated the role of Sestrin2 in the RANKL-RANK-TRAF6 signaling pathway during osteoclast differentiation. Deletion of Sestrin2 (Sesn2) increased bone mass and reduced the number of multinucleated osteoclasts on bone surfaces. RANKL-induced osteoclast differentiation and function decreased in Sesn2 knockout (KO) bone marrow-derived monocytes/macrophages (BMMs) due to inhibition of NFATc1 expression, but osteoblastogenesis was not affected. mRNA expression of RANKL-induced specific osteoclastogenic genes and MAPK protein expression were lower in Sesn2 KO BMMs than wild-type (WT) BMMs after RANKL treatment. However, the Sesn2 deletion did not affect ROS generation or intracellular Ca2+ oscillations during osteoclastogenesis. In contrast, the interaction between TRAF6 and p62 was reduced during osteoclasts differentiation in Sesn2 KO BMMs. The reduction in the TRAF6/p62 interaction and TRAP activity in osteoclastogenesis in Sesn2 KO BMMs was recovered to the WT level upon expression of Flag-Sesn2 in Sesn2 KO BMMs. These results suggest that Sestrin2 has a novel role in bone homeostasis and osteoclasts differentiation through regulation of NFATc1 and the TRAF6/p62 interaction.

Microfluidic confinement enhances phenotype and function of hepatocyte spheroids.

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into three-dimensional (3-D) spheroids is one well-accepted method for extending epithelial phenotype of these cells. Our laboratory has previously observed enhanced function of two-dimensional (2-D, monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study, we wanted to test a hypothesis that culturing hepatocyte spheroids (3-D) in microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of hepatocytes into spheroids with individual diameters of ~100 µm. The hepatocyte spheroids were found to be more functional, as evidenced by higher level of albumin synthesis, bile acid production, and hepatic enzyme expression, in low-volume compared with large-volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. Although decay of hepatic function (albumin secretion) was observed over the course 3 wk, this behavior could be abrogated by inhibiting TGF-ß1 signaling. With TGF-ß1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of 4 wk. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin-22: a potential therapy for alcoholic hepatitis.

CT-like MRI using the zero-TE technique for osseous changes of the TMJ.

OBJECTIVE: This study was conducted to assess the clinical usability of the zero-echo time (ZTE) technique of MRI for evaluating bone changes of the temporomandibular joint (TMJ) in comparison with CBCT. METHODS: Twenty patients with TMJ disorder who underwent both CBCT and MRI were randomly selected. CBCT images were obtained with an Alphard 3030 device (Asahi Roentgen Ind., Co. Ltd, Kyoto, Japan). MRIs were obtained using a 3.0 T scanner (Pioneer; GE Healthcare, Waukesha, WI, USA) and a 21-channel head coil. An isotropic three-dimensional proton-density-weighted ZTE sequence was acquired. Two radiologists evaluated 40 joints of 20 patients for the presence of the following osseous changes: flattening, erosion, osteophyte and sclerosis of the condyle; and flattening, erosion and sclerosis of the articular fossa. CBCT and ZTE-MRI assessments were performed at a 2-month interval. The prevalence-adjusted and bias-adjusted κ statistic was used to analyse interexaminer and intraexaminer agreement and the agreement between ZTE-MRI and CBCT. RESULTS: Intraexaminer and interexaminer agreement analyses of ZTE-MRI showed high reproducibility (κ>0.80), which was comparable to that of CBCT. Flattening, osteophyte and sclerosis of the condyle and all types of bone changes in the mandibular fossa showed nearly perfect agreement between CBCT and ZTE-MRI (κ = 0.80-0.90). Erosion of the condyle showed substantial agreement between both sets of images (κ = 0.65-0.70). CONCLUSIONS: It is suggested that ZTE-MRI provides clinically reliable images for bone assessment in TMJ disorder. MRI may become a beneficial diagnostic tool for patients with both TMJ disc and bone pathology, with advantages involving medical costs and radiation dose.

Assessment of tooth wear based on autofluorescence properties measured using the QLF technology in vitro.

BACKGROUND: The difference in autofluorescence between enamel and dentine layer has prompted recommendations to use the quantitative light-induced fluorescence (QLF) method for quantifying tooth wear (TW). This study investigated the potential of QLF for distinguishing the severity of occlusal TW based on differences in the autofluorescence intensity. METHODS: In total, 106 extracted permanent molars and premolars having suspected wear without pulp exposure were used. The severity of wear was determined by visually examining all teeth using the tooth wear index (TWI) of Smith and Knight. QLF images were captured and converted into 8-bit grayscale images. The difference in the fluorescence intensity (ΔG) was calculated by comparing mean grayscale levels between sound and worn areas. Finally, histological examination was conducted by stereomicroscope to confirm the presence of dentine exposure. RESULTS: 100 teeth were included in the final analysis without six teeth having enamel cracks around worn area. The ΔG values increased with the severity of TW as quantified using conventional TWI codes, and differed significantly between the sound and enamel- and dentine-wear teeth (P < 0.001). The histology indicated that enamel remained on 57 teeth, while 43 teeth had dentine-exposed wear and showed significant differences in ΔG compared with enamel-remained teeth. CONCLUSIONS: The fluorescence intensity differed significantly depending on the presence of dentine exposure. ΔG could be used to distinguish between sound and enamel- and dentine-wear teeth with a significant correlation. These findings indicate that QLF could be useful for determining the severity of TW of occlusal surfaces noninvasively.

Particle-in-Particle Platform for Nanoconfinement-Induced Oncothermia.

Oncothermia, a special form of hyperthermia for oncological purposes, has been widely shown to be an effective mode of cancer therapy. However, its adoption among standard therapeutic practices has been limited by constraints in delivering sufficient thermal energy to tumor targets. To overcome these unique challenges in delivery presented by oncothermic therapeutics, we engineered a novel universal platform for hyperthermia cancer therapy utilizing versatile biocompatible materials. Herein, we show that Gold particle-in-particle (PIP), in which gold nanoparticles are physically confined within PLGA-PEG nanoparticles, significantly enhances thermal energy production by red-shifting the gold nanoparticle's absorption spectra via a mechanism in which we call nanoconfinement-induced therapeutic enhancement (NITE). NITE mediated Gold PIPs significantly suppress breast, skin, and multidrug resistant tumors and result in a multifold increase of heat shock protein expressed by cancer cells in vivo. Cotreatment of Gold PIP with doxorubicin shows a synergistic advantage. By using tumor-bearing mice, significant suppression of tumor growth by Gold PIPs shows the advantage of NITE mediated hyperthermia. Thus, we conclude that NITE mediated Gold PIP can be a strong anticancer therapy because of its sufficient amount of heat generation.

Improving Tumor Specificity and Anticancer Activity of Dasatinib by Dual-Targeted Polymeric Micelles.

To improve tumor targetability and drug efficacy and decrease drug resistance of dasatinib (DSB), the multifunctional micellar nanoparticles that combined the matrix metalloproteinase 2 (MMP2)-sensitive tumor (site) targeting with folate receptor-mediated tumor (cell) targeting were developed. Two major functional polymers, polyethylene glycol (5000 Da)-MMP2-sensitive peptide-phosphoethanolamine (PEG5k-pp-PE) and folic acid-polyethylene glycol (2000 Da)-phosphoethanolamine (FA-PEG2k-PE), were synthesized to construct the dual-targeted micellar nanoparticles (MMP/FR micelles). In the absence of MMP2, the FA was shielded by PEG5k and the MMP/FR micelles showed low bioactivity. In the presence of MMP2, the nanoparticulate structure, stability, and cargo release profile of the MMP/FR micelles were not significantly affected, however, the MMP2-mediated PEG5k deshielding and FA exposure remarkably increased the cellular uptake and anticancer activity of the micelles in the MMP2 and FR expressing (MMP2+/FR+) cells, including multidrug resistant (MDR) cancer cells, rather than the MMP2- and FR- cells. In the 3D MDR tumor spheroids, the significant MMP2-dependent tissue penetration, uptake and cytotoxicity of the MMP/FR micelles were also observed. Furthermore, in the in vivo biodistribution study, the MMP2 and FR dual targeting strategy could significantly prolong the systemic circulation, decrease the nonspecific distribution in nontumor tissues, and increase the tumor accumulation of the polymeric micelles in a melanoma xenograft mouse model. The MMP2-sensitive FR-targeted micelles might have great potential as a tumor-targeted platform for delivery of molecular targeted therapeutics.

Multi-layered nanogels with MMP-sheddable PEG masks: Preparation and promotion of tumor cell uptake by controlling surface characteristics.

We describe a facile and effective poly(ethylene glycol) (PEG) coating method that has not been used previously for decoration of nanogels. Layer-by-layer (LbL) assembly was the technique of choice to introduce PEG onto the surface of multi-layered nanogels (MLNGs). Pegylated MLNGs were prepared simply by sequentially dispersing nanogels in solutions of cationic polymer, anionic polymer, and modified PEGs, and each coating step was validated by measuring changes in size and surface charge. Particularly, a peptide linker that is cleavable by matrix metalloproteinase-2 (MMP-2) in the tumor microenvironment was introduced between PEG layer and nanogels because it is known that PEG reduces the uptake of nanoparticles in tumor cells due to its high mobility. In vitro cell studies demonstrated that MLNGs with MMP-cleavable PEGs enhanced the particle uptake up to 3 times in tumor cells due to unmasking of PEG brushes as compared to pegylated MLNGs without MMP-sensitive peptide linkers.

Plaque autofluorescence as potential diagnostic targets for oral malodor.

The aim of this study was to determine whether the degree of tongue and interdental plaque can be used to assess oral malodor by quantifying their fluorescence as detected using quantitative light-induced fluorescence (QLF) technology. Ninety-nine subjects who complained of oral malodor were included. The level of oral malodor was quantified using the organoleptic score (OLS) and the concentration of volatile sulfur compounds (VSCs). The fluorescence properties of tongue and interdental plaque were quantified as scores calculated by multiplying the intensity and area of fluorescence in QLF-digital images, and the combined plaque fluorescence (CPF) score was obtained by summing the scores for the two regions. The associations of the scores with malodor levels and the diagnostic accuracy of the CPF score were analyzed. The two plaque fluorescence scores and their combined score differed significantly with the level of oral malodor (p<0.001). The CPF score was moderately correlated with OLS (r=0.64) and VSC levels (r=0.54), and its area under the receiver operating characteristic curve was 0.77 for identifying subjects with definite oral malodor (OLS≥2). In conclusion, plaque fluorescence from tongue and interdental sites as detected using QLF technology can be used to assess the level of oral malodor.

Hierarchical self-assembly of magnetic nanoclusters for theranostics: Tunable size, enhanced magnetic resonance imagability, and controlled and targeted drug delivery.

Nanoparticle-based imaging and therapy are of interest for theranostic nanomedicine. In particular, superparamagnetic iron oxide (SPIO) nanoparticles (NPs) have attracted much attention in cancer imaging, diagnostics, and treatment because of their superior imagability and biocompatibility (approved by the Food and Drug Administration). Here, we developed SPIO nanoparticles (NPs) that self-assembled into magnetic nanoclusters (SAMNs) in aqueous environments as a theranostic nano-system. To generate multi-functional SPIO NPs, we covalently conjugated ß-cyclodextrin (ß-CD) to SPIO NPs using metal-adhesive dopamine groups. Polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the ß-CD cavity through high affinity complexation. The core-shell structure of the magnetic nanoclusters was elucidated based on the condensed SPIO core and a PEG shell using electron microscopy and the composition was analyzed by thermogravimetric analysis (TGA). Our results indicate that nanocluster size could be readily controlled by changing the SPIO/PEG ratio in the assemblies. Interestingly, we observed a significant enhancement in magnetic resonance contrast due to the large cluster size and dense iron oxide core. In addition, tethering a tumor-targeting peptide to the SAMNs enhanced their uptake into tumor cells. PTX was efficiently loaded into ß-CDs and released in a controlled manner when exposed to competitive guest molecules. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy. STATEMENT OF SIGNIFICANCE: In this study, we developed multi-functional SPIO NPs that self-assembled into magnetic nanoclusters (SAMNs) in aqueous conditions as a theranostic nano-system. The beta-cyclodextrin (ß-CD) was immobilized on the surfaces of SPIO NPs and RGD-conjugated polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the ß-CD cavity through high affinity complexation. We found that nanocluster size could be readily controlled by varying the SPIO/PEG ratio in the assemblies, and also demonstrated significant improvement of the functional nanoparticles for theranostic systems; enhanced magnetic resonance, improved cellular uptake, and efficient PTX loading and sustained release at the desired time point. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy.

Clinical assessment of oral malodor using autofluorescence of tongue coating.

OBJECTIVES: The aim of this study was to evaluate whether a new method using quantitative light-induced fluorescence-digital (QLF-D) was appropriate for the diagnosis of oral malodor by quantifying the fluorescence of tongue coating. METHODS: This study examined 103 healthy subjects who have an oral malodor as a main complaint. The levels of oral malodor were measured by organoleptic scores (OLS) and volatile sulfur compound (VSC) levels. The fluorescent tongue coating images captured by QLF-D were quantified as the integrated fluorescence score (IF score) by multiplying the intensity and area of fluorescence. The correlations between the fluorescence parameters and OLS as well as VSC levels and the diagnostic accuracy of the IF score were evaluated. RESULTS: The IF score of tongue coating showed a significant positive correlation with the OLS (r=0.54, p<0.01) and the VSC levels (r=0.49, p<0.01). This score was significantly differed with the level of oral malodor (p<0.001), and its AUC was 0.72 in identifying the patient with definite oral malodor (≥OLS 2). CONCLUSIONS: A new method quantifying tongue coating fluorescence detected by QLF-D can be used to diagnose oral malodor and assess its severity in the clinical practice.

Targeted doxorubicin nanotherapy strongly suppressing growth of multidrug resistant tumor in mice.

The rational design of nanomedicine to treat multidrug resistant (MDR) tumors in vivo is described in the study. We prepared multifunctionalized Pluronic micelles that are already well-established to be responsive to low pH and redox in order to systemically deliver doxorubicin (DOX) to MDR tumors. Folic acids (FAs) were introduced on the micelle surface as tumor-targeting molecules. In vitro, the DOX-loaded micelles exerted high cytotoxicity in the DOX-resistant cells by bypassing MDR efflux. Cellular uptake studies clearly demonstrated that FA-conjugated DOX micelles (FA/DOX micelles) were efficiently internalized and accumulated in the MDR cells. In vivo studies indicated significant efficacy of FA/DOX micelles for MDR tumors in mice, and that the volume of tumors was 3 times smaller in this group than that of tumors in the free DOX group, and 8 times smaller than the tumors in the saline group. To the best of our knowledge, this methodology has been recognized to have significantly high efficacy, compared to previously reported DOX nanoparticle formulations. This superior anti-tumor efficacy of FA/DOX micelles in MDR tumor-bearing mice can be attributed to FA-targeted and -mediated endocytosis, inhibition of MDR effect, and subsequent DOX release triggered by dual stimuli (low pH and redox) inside the tumor. Given the promise of the multifunctional micelle mediated delivery on inhibition of MDR tumor growth, FA/DOX micelle platform is a much sought after goal for cancer chemotherapy, especially for cancers resistant to anticancer drugs.

Ca(2+) is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line.

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca(2+) may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca(2+) concentration ([Ca(2+)]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca(2+)]i increase with nonspecific Ca(2+) channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca(2+)]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca(2+) chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca(2+) may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca(2+) regulation of the WNK-OSR1 pathway in intact cells.

Horseradish peroxidase-catalysed in situ-forming hydrogels for tissue-engineering applications.

In situ-forming hydrogels are an attractive class of implantable biomaterials that are used for biomedical applications. These injectable hydrogels are versatile and provide a convenient platform for delivering cells and drugs via minimally invasive surgery. Although several crosslinking methods for preparing in situ forming hydrogels have been developed over the past two decades, most hydrogels are not sufficiently versatile for use in a wide variety of tissue-engineering applications. In recent years, enzyme-catalysed crosslinking approaches have been emerged as a new approach for developing in situ-forming hydrogels. In particular, the horseradish peroxidase (HRP)-catalysed crosslinking approach has received increasing interest, due to its highly improved and tunable capacity to obtain hydrogels with desirable properties. The HRP-catalysed crosslinking reaction immediately occurs upon mixing phenol-rich polymers with HRP and hydrogen peroxide (H2O2) in aqueous media. Based on this unique gel-forming feature, recent studies have shown that various properties of formed hydrogels, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H2O2. In this review, we outline the versatile properties of HRP-catalysed in situ-forming hydrogels, with a brief introduction to the crosslinking mechanisms involved. In addition, the recent biomedical applications of HRP-catalysed in situ-forming hydrogels for tissue regeneration are described.

In situ formation of enzyme-free hydrogels via ferromagnetic microbead-assisted enzymatic cross-linking.

In situ formation of horseradish peroxidase (HRP)-free gelatin hydrogels was achieved via ferromagnetic microbead-assisted enzymatic cross-linking. Gelation time and mechanical stiffness of the hydrogels can be tuned in situ, which makes HRP-free gelatin hydrogels suitable for injectable cell delivery.

Macro/Nano-gel composite as an injectable and bioactive bulking material for the treatment of urinary incontinence.

Many women around the world are suffering from urinary incontinence, defined as the unintentional leakage of urine by external abnormal pressure. Although various kinds of materials have been utilized to treat this disease, therapies that are more effective are still needed for the treatment of urinary incontinence. Here, we present a macro/nanogel composed of in situ forming gelatin-based macrogels and self-assembled heparin-based nanogels, which can serve as an injectable and bioactive bulking material for the treatment of urinary incontinence. The hybrid hydrogels were prepared via enzymatic reaction in the presence of horseradish peroxidase and hydrogen peroxide. Incorporating a growth factor (GF)-loaded heparin nanogel into a gelatin gel matrix enabled the hybrid gel matrix to release GF continuously up to 28 days. Moreover, we demonstrated that the hydrogel composites stimulated the regeneration of the urethral muscle tissue surrounding the urethral wall and promoted the recovery of their biological function when injected in vivo. Thus, the macro/nanohydrogels may provide an advanced therapeutic technique for the treatment of urinary incontinence as well as an application for regenerative medicine.

Differentiation of endothelial progenitor cells into endothelial cells by heparin-modified supramolecular pluronic nanogels encapsulating bFGF and complexed with VEGF165 genes.

Specific genes and growth factors are involved in stem cell differentiation. In this study, we fabricated a delivery carrier for both protein and gene delivery that was introduced into human endothelial progenitor cells (EPCs). The highly negative charge carried by the heparin-modified pluronic nanogels allowed for binding to growth factors and localization in the core of nanogels. The residues of negatively charged heparin can complex with positively charged cationic materials, making it suitable for gene delivery. Supramolecular nanogels can be easily encapsulated the hydrophilic drugs and highly positive surfaces can be complexed with negative charge carrying plasmid DNA (pDNA). The size distribution, gel retardation, and denaturation of encapsulated growth factors and supramolecular nanogels modified with heparin were evaluated. The supramolecular nanogels containing basic fibroblast growth factors and complexing VEGF165 pDNA internalized into EPCs have been well formed vascular formation in matrigel gels. Proteins and genes introduced into EPCs using nanogels promoted neovascularization in an animal model of limb ischemia. EPCs that differentiated into endothelial cells both in vitro and in vivo were tested.